[Nhcoll-l] [EXTERN] Re: Best practices for ethanol-fixed specimens

Sat Apr 25 08:29:05 EDT 2026

Not explicitly with regards to isotopic analyses (I too struggled and
failed to find suitable literature on this), but rather from results of my
own trials following a tip from my graduate advisory team.  The available
literature is mainly wity regards to protein degradation in general when
tissue is preserved in formalin or ethanol. Some of these include:

·       LIANG ET AL 2024. Deep silicification–assisted long-term
preservation of structural and genomic information across biospecies: From
micro to macro. PNAS 124(42). https://doi.org/10.1073/pnas.2408273121

·       E. Sarot, et al 2017. Assessment of alternatives to environmental
toxic formalin for DNA conservation in biological specimens. *Environ. Sci.
Pollut. Res. *24, 16985–16993.

·       S. M. Hykin, et al 2015. A method to recover genomic-scale DNA
sequence data from formalin-fixed museum specimens using high-throughput
sequencing. *PLoS One *10, 16 .

·        Oosting et al. 2020. *DNA degradation in fish: Practical solutions
and guidelines to improve DNA preservation for genomic research. Ecology
and Evolution  10(16).*

*https://doi.org/10.1002/ece3.6558 <https://doi.org/10.1002/ece3.6558>*

On Fri, Apr 24, 2026 at 5:43 PM John E Simmons <simmons.johne at gmail.com>
wrote:

> A correction needs to be made concerning the use of the term
> “pseudo-fixative.”
>
>
>
> Fixation has many meanings, but in general it refers to a chemical or
> physical process that is used to prevent postmortem changes in tissue. From
> a histological point of view, fixation also prepares tissues to be
> receptive to staining, sectioning, or other treatments. There are dozens of
> definitions of fixation in the literature, and dozens of recipes for
> fixatives to produce particular results. Tissues can be fixed by
> dehydration, heat, salts, acids, and other chemicals. Fixation has been
> practiced for a very long time―histologists cite the year 1833 as the date
> of the origin of modern fixation because that was when chromic acid came
> into use as a hardening agent, well before formaldehyde had even been
> discovered, but specimens were being fixed by various means well before the
> 1833. The choice of fixative or fixation process depends on what organism
> you are working with and what results you want.
>
>
>
> As discussed in detail in the second edition of *Fluid Preservation: A
> Comprehensive Reference* (2025) the term pseudo-fixative is both
> incorrect and misleading. It seems to have been coined by Stoddard to
> differentiate between fixatives that form covalent bonds or crosslinks
> (e.g., formaldehyde and gluteraldehyde) and fixatives that do not form
> crosslinks. In fact, there is no such thing as a pseudo-fixative because
> fixation cannot be defined by the formation of crosslinks. There are many
> ways to fix tissue that do not form crosslinks.
>
>
>
> The specimens in question that were put into 95% ethanol have no doubt
> been fixed, and as Dirk has correctly pointed out, that process has no
> doubt resulted in osmotic damage to the tissues. Other treatments can still
> be done on the specimens, including other fixation techniques, but to refer
> to the use of 95% ETOH as a pseudo-fixative is incorrect. Fixation in
> alcohol does, in fact, result in changes in the tissues in that it unwinds
> and disorders proteins and alters the patterns of hydrogen bonding as the
> tissue is dehydrated.
>
> --John
>
> John E. Simmons
> US Fulbright Senior Scholar
> Writer and Museum Consultant, Museologica
> *and*
> Research Associate, Earth and Mineral Sciences Museum & Art Gallery, Penn
> State University
> *and*
> Investigador Asociado, Departamento de Ornitología, Museo de Historia
> Natural, Universidad Nacional Mayor de San Marcos, Lima
>
>
> On Fri, Apr 24, 2026 at 1:43 AM Dirk Neumann <d.neumann at leibniz-lib.de>
> wrote:
>
>> ... and that "once they are stable in 95%", but osmotic damage is done
>> (which usually is irreversible and includes cell rupture, which makes the
>> specimens less suited for histology and analysis of fine cell structures,
>> embryos tend to be used for).
>>
>> With best wishes
>> Dirk
>>
>>
>> Am 23.04.2026 um 23:35 schrieb Simon Moore:
>>
>> ACHTUNG/ATTENTION: Diese E-Mail stammt von einem externen Absender. /
>> This e-mail comes from an external sender.
>>
>>
>> Hi Beth,
>>
>> Remember that EtOH is only a pseudo-fixative as it doesn’t really bring
>> about a chemical change.
>> Sorry to be such a pedant!
>>
>> With all good wishes, Simon
>>
>> Simon Moore MIScT, RSci, FLS, ACR
>> Conservator of Natural Sciences and Cutlery Historian.
>>
>> www.natural-history-conservation.com
>>
>>
>>
>>
>> > On 23 Apr 2026, at 21:30, Elizabeth Wommack <ewommack at uwyo.edu>
>> <ewommack at uwyo.edu> wrote:
>> >
>> > Hi Verity,
>> >
>> > One thing you could do is check to see if they have finished fixing in
>> the 95% ETOH. Check to see if the concentration of the ETOH they are in is
>> 95%. If it is less then they may still be exchanging water with the
>> preservative, and you might want to put them in some fresh 95% ETOH for a
>> bit longer. Once they are stable at 95% ETOH, I switch ETOH fixed material
>> to 70% ETOH for long-term storage.
>> >
>> > cheers,
>> > Beth
>> >
>> > Elizabeth Wommack, PhD
>> > Curator and Collections Manager of Vertebrates
>> > University of Wyoming Museum of Vertebrates
>> > Berry Biodiversity Conservation Center
>> > University of Wyoming,
>> > Laramie, WY 82071
>> > ewommack at uwyo.edu
>> > pronouns: she, her, herself
>> > www.uwymv.org
>> > UWYMV Collection Use PolicyFrom: Nhcoll-l
>> <nhcoll-l-bounces at mailman.yale.edu> <nhcoll-l-bounces at mailman.yale.edu>
>> on behalf of Dirk Neumann <d.neumann at leibniz-lib.de>
>> <d.neumann at leibniz-lib.de>
>> > Sent: Thursday, April 23, 2026 00:09
>> > To: nhcoll-l at mailman.yale.edu <nhcoll-l at mailman.yale.edu>
>> <nhcoll-l at mailman.yale.edu>
>> > Subject: Re: [Nhcoll-l] [EXTERN] Re: Best practices for ethanol-fixed
>> specimens
>> >  ◆ This message was sent from a non-UWYO address. Please exercise
>> caution when clicking links or opening attachments from external sources.
>> >
>> > Hi Verity,
>> >
>> > I would opt for 70%; 95% not only strongly dehydrates the specimens,
>> but also causes a lot of osmotic pressure on tissue membranes, which might
>> effect the usefulness of the specimens for morphological analysis. Tissue
>> subsampling and subsequent storage of tissues in 95% for later molecular
>> investigation might be better.
>> >
>> > Hope this helps
>> > Dirk
>> >
>> >
>> > Am 22.04.2026 um 22:50 schrieb Shoobs, Nate:
>> >>
>> >>
>> >> Depends on what you want to use them for!
>> >> Formalin is not a great long-term preservative, even though it's a
>> great fixative. Since the tissue is fixed already, unless you want to
>> post-fix for some specific purpose like histology, I'd leave them in
>> ethanol.
>> >> It's fine to keep things in 95% indefinitely, but if you think they
>> will be used mainly for anatomical study, swapping the 95% for 70-75%  is
>> better for long term storage and will keep the tissues flexible. Some
>> institutions use 80% as a compromise between genetic and anatomical
>> preservation. If you typically voucher frozen material, I'd recommend
>> taking your tissue samples and freezing them before stepping the lot(s)
>> down in concentration. You could even chuck a whole individual in 95% into
>> the freezer.
>> >>
>> >> Personally, I rarely if ever use graded ethanol series for going from
>> high to lower concentration, especially for fresher material, I only bother
>> when going from low-high, but I'm unsure how vertebrates typically handle.
>> >> My 2 cents!
>> >> -Nate
>> >> --<image001[26].png>
>> >>
>> >> Nathaniel F. Shoobs
>> >> Curator of Mollusks
>> >> College of Arts & Sciences Dept. of Evolution, Ecology, and Organismal
>> Biology
>> >> Museum of Biological Diversity, 1315 Kinnear Rd, Columbus, OH 43212
>> >> 614-688-1342 (Office)
>> >> mbd.osu.edu/collections/invertebrates
>> >> From: Nhcoll-l <nhcoll-l-bounces at mailman.yale.edu>
>> <nhcoll-l-bounces at mailman.yale.edu> on behalf of Mathis,Verity L
>> <vmathis at flmnh.ufl.edu> <vmathis at flmnh.ufl.edu>
>> >> Date: Wednesday, April 22, 2026 at 4:38 PM
>> >> To: nhcoll-l at mailman.yale.edu <nhcoll-l at mailman.yale.edu>
>> <nhcoll-l at mailman.yale.edu>
>> >> Subject: [Nhcoll-l] Best practices for ethanol-fixed specimens
>> >>
>> >> This Message Is From an External Sender
>> >> This message came from outside your organization.
>> >> Report Suspicious
>> >>  Hello all
>> >> I am hoping to get feedback on the best practices for some recently
>> fixed fluid specimens.  During some recent field work, our lab fixed some
>> rodent fetuses in 95% ethanol while prepping.  I’m hoping for some advice
>> on how best store them long-term now that they are back in the
>> lab/collections.  Do I keep them in 95% ETOH, re-fix them in formalin, or
>> step them down to 70% ETOH for long-term storage?  Or something else
>> altogether?
>> >>  Thanks for any insight!
>> >> Verity
>> >> ******************************
>> >> Verity L. Mathis, Ph.D.
>> >> Mammal Collections Manager
>> >> Florida Museum of Natural History
>> >> University of Florida
>> >> 1659 Museum Road
>> >> Gainesville FL 32611
>> >> Phone: (352) 273-2114
>> >> Email: vmathis at flmnh.ufl.edu
>> >> Website: https://www.floridamuseum.ufl.edu/mammals/
>> >> FM Mammal Collection Google Scholar: https://tinyurl.com/flmnh-mammals
>> >> ᘛ⁐̤ᕐᐷ Support Florida Museum Mammalogy ᘛ⁐̤ᕐᐷ
>> >>
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>> >>
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>> >> NHCOLL-L is brought to you by the Society for the Preservation of
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>> >
>> > --
>> > ****
>> >  Dirk Neumann
>> > Collection Manager, Hamburg
>> >  Postal address:
>> > Museum of Nature Hamburg
>> > Leibniz Institute for the Analysis
>> > of Biodiversity Change
>> > Dirk Neumann
>> > Martin-Luther-King-Platz 3
>> > 20146 Hamburg
>> > +49 40 238 317 – 628
>> > d.neumann at leibniz-lib.de
>> > www.leibniz-lib.de
>> >  --
>> > Stiftung Leibniz-Institut zur Analyse des Biodiversitätswandels
>> > Postanschrift: Adenauerallee 127, 53113 Bonn, Germany
>> >
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>> >
>> > --
>> > Stiftung Leibniz-Institut zur Analyse des Biodiversitätswandels
>> > Postanschrift: Adenauerallee 127, 53113 Bonn, Germany
>> >
>> > Stiftung des öffentlichen Rechts;
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>> Grüter (Kaufm. Geschäftsführer)
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>> >
>> > _______________________________________________
>> > NHCOLL-L is brought to you by the Society for the Preservation of
>> > Natural History Collections (SPNHC), an international society whose
>> > mission is to improve the preservation, conservation and management of
>> > natural history collections to ensure their continuing value to
>> > society. See http://www.spnhc.org for membership information.
>> > Advertising on NH-COLL-L is inappropriate.
>>
>>
>>
>> --
>>
>> ******
>>
>>
>>
>> *Dirk Neumann*
>>
>> Collection Manager, Hamburg
>>
>>
>>
>> Postal address:
>>
>> *Museum of Nature Hamburg*
>> Leibniz Institute for the Analysis
>>
>> of Biodiversity Change
>>
>> Dirk Neumann
>>
>> Martin-Luther-King-Platz 3
>>
>> 20146 Hamburg
>> +49 40 238 317 – 628
>>
>> *d.neumann at leibniz-lib.de <d.neumann at leibniz-lib.de>*
>>
>> www.leibniz-lib.de
>>
>>
>>
>> --
>> Stiftung Leibniz-Institut zur Analyse des Biodiversitätswandels
>> Postanschrift: Adenauerallee 127, 53113 Bonn, Germany
>>
>> Stiftung des öffentlichen Rechts;
>> Generaldirektion: Prof. Dr. Bernhard Misof (Generaldirektor), Adrian
>> Grüter (Kaufm. Geschäftsführer)
>> Sitz der Stiftung: Adenauerallee 160 in Bonn
>> Vorsitzender des Stiftungsrates: Dr. Michael Wappelhorst
>>
>>
>> --
>> Stiftung Leibniz-Institut zur Analyse des Biodiversitätswandels
>> Postanschrift: Adenauerallee 127, 53113 Bonn, Germany
>>
>> Stiftung des öffentlichen Rechts;
>> Generaldirektion: Prof. Dr. Bernhard Misof (Generaldirektor), Adrian
>> Grüter (Kaufm. Geschäftsführer)
>> Sitz der Stiftung: Adenauerallee 160 in Bonn
>> Vorsitzender des Stiftungsrates: Dr. Michael H. Wappelhorst
>> _______________________________________________
>> Nhcoll-l mailing list
>> Nhcoll-l at mailman.yale.edu
>> https://mailman.yale.edu/mailman/listinfo/nhcoll-l
>>
>> _______________________________________________
>> NHCOLL-L is brought to you by the Society for the Preservation of
>> Natural History Collections (SPNHC), an international society whose
>> mission is to improve the preservation, conservation and management of
>> natural history collections to ensure their continuing value to
>> society. See http://www.spnhc.org for membership information.
>> Advertising on NH-COLL-L is inappropriate.
>>
> _______________________________________________
> Nhcoll-l mailing list
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>
> _______________________________________________
> NHCOLL-L is brought to you by the Society for the Preservation of
> Natural History Collections (SPNHC), an international society whose
> mission is to improve the preservation, conservation and management of
> natural history collections to ensure their continuing value to
> society. See http://www.spnhc.org for membership information.
> Advertising on NH-COLL-L is inappropriate.
>
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